Rough Brucella neotomae provides protection against Brucella suis challenge in mice.

Brucellosis is one of the most common zoonotic diseases worldwide. Almost 500,000 new human cases occur each year; yet there is no vaccine for human use. Moreover, there is no universal Brucella vaccine that would provide protection against all pathogenic species of Brucella. We generated a rough, live-attenuated B. neotomae strain by deleting the wboA gene encoding a glycosyltransferase. This strain lacks the O-side chain in its lipopolysaccharide (LPS) and thus the vaccinated animals can be differentiated serologically from the field-infected animals. We tested the efficacy of rough B. neotomae strain to stimulate dendritic cells compared to the smooth wild type strain. Based on TNF-α production, our data suggests that a significantly higher stimulation was obtained when dendritic cells were stimulated with the rough vaccine strain compared to the smooth wild type B. neotomae. Furthermore, the rough mutant was cleared from mice within 6 weeks even at a dose as high as 2 x 108 CFU. Vaccinated mice showed significantly higher level of protection against a virulent B. suis 1330 challenge compared to the control mice. Antibody titers in the mice and cytokine production by the splenocytes from the vaccinated mice showed a Th1 mediated immune response that correlated with the protection.

Design and influence of gamma-irradiation on the biopharmaceutical properties of nanoparticles containing an antigenic complex from Brucella ovis.

Despite vaccination campaigns, brucellosis is still one of the most common bacterial zoonosis in the world. This work describes the development of a novel formulation strategy to the delivery of the Brucella ovis antigenic extract (HS) into ovine mucosal surfaces. Thus, HS was entrapped in conventional and mannosylated poly(anhydride) nanoparticles by the solvent displacement method, and the resulting nanosystems were gamma-irradiated to accomplish the sterilization required for the ophthalmic administration route. Sterilization, at either 10 kGy or 25 kGy, did not modify the size, morphology and antigen content of the nanoparticles. Similarly, the integrity and antigenicity of the entrapped antigen were not affected by gamma-irradiation. The 25 kGy gamma-irradiation dose seemed to influence negatively the HS release from the carriers. However, and in accordance with the Pearson's correlation, all the release patterns followed a similar tendency. Furthermore, the stability of the vaccine systems on lachrymal and nasal ovine fluids, showed that gamma-irradiation had no significant effects on the vaccine systems. Since all the vaccine systems accomplished the pharmacopoeial biological tests required for gamma-irradiation doses under 25 kGy, these results are highly suggestive for the use of HS loaded poly(anhydride) nanoparticles as an efficient vaccine delivery system for brucellosis immunoprophylaxis, especially for ophthalmic administration.

The Brucella abortus S19 DeltavjbR live vaccine candidate is safer than S19 and confers protection against wild-type challenge in BALB/c mice when delivered in a sustained-release vehicle.

Brucellosis is an important zoonotic disease of nearly worldwide distribution. Despite the availability of live vaccine strains for bovine (S19, RB51) and small ruminants (Rev-1), these vaccines have several drawbacks, including residual virulence for animals and humans. Safe and efficacious immunization systems are therefore needed to overcome these disadvantages. A vjbR knockout was generated in the S19 vaccine and investigated for its potential use as an improved vaccine candidate. Vaccination with a sustained-release vehicle to enhance vaccination efficacy was evaluated utilizing the live S19 DeltavjbR::Kan in encapsulated alginate microspheres containing a nonimmunogenic eggshell precursor protein of the parasite Fasciola hepatica (vitelline protein B). BALB/c mice were immunized intraperitoneally with either encapsulated or nonencapsulated S19 DeltavjbR::Kan at a dose of 1 x 10(5) CFU per animal to evaluate immunogenicity, safety, and protective efficacy. Humoral responses postvaccination indicate that the vaccine candidate was able to elicit an anti-Brucella-specific immunoglobulin G response even when the vaccine was administered in an encapsulated format. The safety was revealed by the absence of splenomegaly in mice that were inoculated with the mutant. Finally, a single dose with the encapsulated mutant conferred higher levels of protection compared to the nonencapsulated vaccine. These results suggest that S19 DeltavjbR::Kan is safer than S19, induces protection in mice, and should be considered as a vaccine candidate when administered in a sustained-release manner.

[Experimental evaluation of the brucellosis therapeutic vaccine efficacy].

Use of antibiotics can't completely solve the problem of brucellosis treatment, especially its chronic forms, because antibacterial preparations do not eliminate main pathogenetic factor of the disease--sensibilization of the macroorganism. It makes actual the question about complex immuno- and antibacterial therapy. Long-term clinical experience proved high effectiveness of a therapeutic brucellosis vaccine. Earlier this preparation was manufactured in Research Institute of Vaccines and Sera in Tbilisi (Georgia). To date new composition of components of the vaccine has been developed, and manufacturing and control methods have been improved. Marked desensitizing effect of the vaccine and its stimulatory action on cellular and humoral immunity has been observed. In 2002 technological normative documentation for manufacturing and use of the vaccine was developed in the Research Institute of Microbiology (Kirov) and production of the vaccine began.

Molecular host-pathogen interaction in brucellosis: current understanding and future approaches to vaccine development for mice and humans.

Brucellosis caused by Brucella spp. is a major zoonotic disease. Control of brucellosis in agricultural animals is a prerequisite for the prevention of this disease in human beings. Recently, Brucella melitensis was declared by the Centers for Disease Control and Prevention to be one of three major bioterrorist agents due to the expense required for the treatment of human brucellosis patients. Also, the economic agricultural loss due to bovine brucellosis emphasizes the financial impact of brucellosis in society. Thus, vaccination might efficiently solve this disease. Currently, B. abortus RB51 and B. melitensis REV.1 are used to immunize cattle and to immunize goats and sheep, respectively, in many countries. However, these genetically undefined strains still induce abortion and persistent infection, raising questions of safety and efficiency. In fact, the REV.1 vaccine is quite virulent and apparently unstable, creating the need for improved vaccines for B. melitensis. In addition, Brucella spp. may or may not provide cross-protection against infection by heterologous Brucella species, hampering the acceleration of vaccine development. This review provides our current understanding of Brucella pathogenesis and host immunity for the development of genetically defined efficient vaccine strains. Additionally, conditions required for an effective Brucella vaccine strain as well as the future research direction needed to investigate Brucella pathogenesis and host immunity are postulated.

Statistical procedures for calculating the residual virulence of Brucella abortus strain 19 (S19) and Brucella melitensis strain Rev 1 vaccines in mice: theoretical basis and practical applications.

Regular control of the biological quality of live Brucella abortus strain 19 (S19) and B. melitensis strain Rev 1 vaccines is essential for the successful management of ruminant brucellosis in affected countries. The reference procedures recommended by the OIE (World organisation for animal health) and the European Pharmacopoeia include the determination of residual virulence, expressed as the recovery time 50 (RT50), of the tested (problem) vaccine in a reference mouse model compared with the RT50 of the corresponding reference strains in the same assay. The underlying statistical procedure applied is based on a parallel line assay and a classical probit model. In practice, the currently recommended procedure for calculating the RT50 is based on a graphical method which has never been described in detail. This paper provides a full description of this graphical method with the aim of making the technique comprehensible and accessible to all interested biologists. The procedure is somewhat cumbersome and very few laboratories apply the OIE and European Pharmacopoeia recommendations on a regular basis. Moreover, since this reference graphical method shows some statistical inconsistencies, a dedicated internet interface has been developed to perform RT50 calculations and is now available free of charge on the web (www.afssa.fr/interne/rev2.html).

Safety of Brucella abortus strain RB51 vaccine in non-target ungulates and coyotes.

Brucellosis is endemic in free-ranging elk (Cervus elaphus) and bison (Bison bison) in the Greater Yellowstone Area (GYA; USA). It is possible that an oral brucellosis vaccine could be developed and disseminated in the GYA to reduce disease transmission. Should this occur, non-target species other than elk and bison may come in contact with the vaccine resulting in morbidity or mortality. To assess biosafety, bighorn sheep (Ovis canadensis; n = 10), pronghorn (Antilocapra americana; n = 9), mule deer (Odocoileus hemionus; n = 11), moose (Alces alces shirasi; n = 10), and coyotes (Canis latrans; n = 24) were given a single oral dose of at least 1.0 x 10(10) colony-forming units of Brucella abortus strain RB51 vaccine (RB51). Animals were randomly divided into vaccinated and control groups. Ungulates were captured, blood sampled, and swabs taken from the nares, rectum, and vagina for bacterial culture on day 0, 42, and 84 post-inoculation (PI). On day 42, the vaccinated group became a control group and vice versa in a crossover design. Blood and swab samples were taken from coyotes on days 0, 14, 28, and 42 PI. There was no crossover for the coyote study. Two coyotes from each group were also euthanized and cultured for RB51 on days 42, 84, 168, and 336 PI. Blood samples were analyzed for hematologic changes and antibodies to RB51 using a modified dot-blot assay. No morbidity or mortality as a result of vaccination was observed in any animal. There were no differences in hematologic parameters at any time for ungulate species; vaccinated coyotes had higher hematocrit, hemoglobin, and eosinophil counts (P < or = 0.006). All individuals, except some moose, seroconverted to RB51. Strain RB51 was cultured from oropharyngeal lymph nodes from one coyote 42 days PI and from a moose 117 days PI. This study suggested that a single oral dose of RB51 was safe in these species.

Comparison between immune responses and resistance induced in BALB/c mice vaccinated with RB51 and Rev. 1 vaccines and challenged with Brucella melitensis bv. 3.

BALB/c mice were immunized with live rough Brucella abortus RB51 or smooth Brucella melitensis Rev. 1 vaccines and challenged with a B. melitensis field strain. Protection was assessed by a variety of serological tests and recovery of vaccinal and challenge strains by culture. Mice vaccinated with RB51 gave negative results in the conventional serological tests prior to challenge, namely; standard tube agglutination test (SAT), Rose Bengal plate test (RBPT), buffered acidified plate antigen test (BAPAT), and mercaptoethanol test (MET). Sero-conversion took place to a whole-cell bacterial buffered RB51 antigen after vaccination and persisted for 7 weeks post-vaccination. Mice challenged with B. melitensis were assessed for bacterial load and immune response for 12 weeks after challenge. Protection units were showed that Rev. 1 vaccine was superior to RB51 vaccine in protection of mice against B. melitensis. However, RB51 vaccine has the advantage that it would not elicit antibodies to standard serological tests based on the LPS O antigen. RB51 vaccine could therefore be used for control of B. melitensis infection and avoid confusion in the use of standard sero-diagnostic tests.

Comparison of the efficacy of Brucella abortus strain RB51 and Brucella melitensis Rev. 1 live vaccines against experimental infection with Brucella melitensis in pregnant ewes.

To test the efficacy of rough Brucella strain vaccines in sheep, a vaccine recently developed in cattle (Brucella abortus strain RB51) was assessed in comparison with the conventional Rev. 1 vaccine. Forty-five ewes from twelve to fourteen months of age, from brucellosis-free flocks, were allotted to three groups of fifteen ewes each. Group one was vaccinated by the conjunctival route with 1.73 x 10(8) colony forming units (CFU) of Rev. 1 vaccine. Group two was vaccinated subcutaneously with 11 x 10(9) CFU of RB51 vaccine and group three was considered as a control. All sheep were challenged at two to three months of gestation with 5 x 10(7) CFU of virulent B. melitensis H38. Vaccination with RB51 vaccine did not result in the production of any antibodies against the O-side chain of lipopolysaccharide, as measured by conventional serological tests (Rose Bengal plate test and complement fixation test). Protection of sheep against abortion and excretion of virulent Brucella strain in vaginal fluid, aborted foetuses and/or non viable lambs at parturition and abortion was significantly lower than that afforded by Rev. 1 vaccine. The difference compared to the control group was not significant. Data from this study suggest that the RB51 vaccine used for cattle vaccination does not provide effective protection of sheep against abortion induced by B. melitensis.

Safety of Brucella abortus strain RB51 in deer mice.

Brucella abortus strain RB51 is an approved brucellosis vaccine for use in cattle that may have potential as an oral vaccine for use in elk (Cervus elaphus) and/or bison (Bison bison). This study was designed to determine effects of strain RB51 on deer mice (Peromyscus maniculatus), a nontarget species that could have access to treated baits in a field situation. In February 1994, 90 mice were orally dosed or intraperitoneally injected with 1 x 10(8) colony forming units strain RB51 and 77 controls were similarly dosed with sterile saline. At weekly intervals through early April 1994, 4 to 6 mice from each group were euthanized, gross necropsies performed, spleens and uteruses cultured, and tissues examined histologically. All orally inoculated mice cleared the infection by 6 wk post-inoculation (PI). While most of the injected mice cleared the infection by 7 wk PI, a few required 9 wk. There were minimal adverse effects attributable to strain RB51. Apparently, strain RB51 would not negatively impact P. maniculatus populations if it were used in a field situation. Also, deer mice appear to be able to clear the vaccine in 6 to 9 wk, thus the probability of these mice transmitting the vaccine to other animals is low.

Safety of Brucella abortus strain RB51 in bull elk.

Some of the elk (Cervus elaphus nelsoni) of the Greater Yellowstone Area (Wyoming, Idaho, Montana; USA) are infected with Brucella abortus, the bacterium that causes bovine brucellosis. Brucella abortus strain RB51 vaccine is being considered as a means to control B. abortus induced abortions in cow elk. However, the most probable vaccination strategies for use in free-ranging elk might also result in some bull elk being inoculated, thus, it is important to insure that the vaccine is safe in these animals. In the winter of 1995, 10 free-ranging bull elk calves were captured, tested for B. abortus antibodies, and intramuscularly inoculated with 1.0 x 10(9) colony forming units (CFU) of B. abortus strain RB51. Blood was collected for hemoculture and serology every 2 wk after inoculation for 14 wk. Beginning 4 mo postinoculation and continuing until 10 mo postinoculation elk were serially euthanized, necropsied, and tissues collected for culture and histopathology. These elk cleared the organism from the blood within 6 wk and from all tissues within 10 mo. No lesions attributable to B. abortus were found grossly and only minimal to mild lymphoplasmacytic epididymitis was found in a few elk on histologic examination. In a separate study, six adult bull elk from Wind Cave National Park (South Dakota, USA) were taken to a ranch near Carrington (North Dakota, USA). Three were orally inoculated with approximately 1.0 x 10(10) CFU of RB51 and three were inoculated with corn syrup and saline. Ninety days post-inoculation semen was examined and cultured from these bulls. Strain RB51 was not cultured from their semen at that time. There were no palpable abnormalities in the genital tract and all elk produced viable sperm. Although they contain small sample sizes, these studies suggest that B. abortus strain RB51 is safe in bull elk.

Safety and efficacy of Brucella abortus strain RB51 vaccine in captive pregnant elk.

Brucella abortus strain RB51 is a laboratory-derived rough mutant of virulent B. abortus strain 2308 used as a vaccine because it induces antibodies that do not react on standard brucellosis serologic tests. Strain RB51 vaccine was evaluated in pregnant captive elk (Cervus elaphus) to determine (1) if it induced abortion and (2) if it protected against abortion following subsequent challenge. The time period of this study (February-June, 1998) was similar to field conditions where elk are vaccinated and possibly exposed to B. abortus. Fourteen elk were randomly and equally divided into vaccinated and control groups. The vaccinated group was vaccinated intramuscularly with 1.03 x 10(10) colony-forming units (CFU) of strain RB51 and seroconverted postvaccination. Antibodies to strain RB51 were detected by a modification of an existing dot-blot assay. Both groups were challenged 40 days postvaccination with 9.8 x 10(6) CFU of B. abortus strain 2308 administered intraconjunctivally. The first abortion occurred 38 days postchallenge. Abortion occurred in all control elk and in five of seven vaccinated elk 5 to 12 wk postchallenge (P = 0.23). Mixed strain RB51 and 2308 infections were present in fetuses and vaginas from the vaccinated group whereas only strain 2308 was cultured from control group fetuses and vaginal swabs. Further evaluation of strain RB51 will be necessary to determine if it will be safe and efficacious in free-ranging pregnant elk.

Safety of Brucella abortus strain RB51 in Bison.

To determine the safety of Brucella abortus strain RB51 (SRB51) vaccine in American bison (Bison bison), 31 animals from a herd with brucellosis were used. In October 1996, 10 adult bison males and seven calves were vaccinated with the standard calfhood cattle dose of 1.8 x 10(10) colony forming units (CFU) of SRB51 subcutaneously while the adult females received the standard adult cattle dose of 1 x 10(9) CFU. Western immunoblot indicated the presence of SRB51 antibodies following vaccination. To evaluate prolonged bacterial colonization of tissues, the adult males, calves, and three adult females were divided into two groups which were slaughtered at either 13 or 16 wk post-vaccination. At necropsy, tissue samples were obtained for B. abortus culture from the liver, spleen, lymph nodes, and reproductive tract of each animal. While B. abortus field strain was cultured from one adult bull, no SRB51 was isolated from any of the animals. Seven pregnant females were monitored until parturition for signs of abortions and fetal lesions. Six cows delivered healthy calves and one delivered a dead full-term calf that was brucellae negative. Based on these results, administration of SRB51 to bison did not cause prolonged bacterial colonization of tissues in calves, adult males, or adult females. Furthermore, SRB51 did not induce abortions following vaccination in the second month of gestation.

Field evaluation of an indirect ELISA for detection of brucellosis in lowland Bolivia.

Bovine brucellosis exists endemically at an estimated prevalence of 10% in the developing dairy industry of Santa Cruz in tropical Bolivia. This paper describes field testing of an FAO/IAEA indirect ELISA for brucellosis, as a possible replacement confirmatory test for the complement fixation test (CFT). The ELISA and CFT were compared on sera from 3 cattle populations: a non-vaccinated negative population, an S19-vaccinated negative population, and a brucellosis-positive population of unknown vaccination status. The CFT and ELISA showed excellent specificities of 100% and 98% respectively against the negative non-vaccinated group. The CFT maintained a specificity of 98% against the S19-vaccinated negative group, but ELISA specificity fell to 83% using a cut-off of 20% of positive control, and 94% using a cut-off of 40% of positive control. Against sera from the positive population, the ELISA gave many more positive reactions than the CFT, probably a combination of both higher sensitivity and lower specificity. It is concluded that as Santa Cruz is entering a phase of brucellosis control rather than eradication, the extra sensitivity of the ELISA is not valuable enough to risk a higher level of false positive reactions, especially as S19 vaccination is being increasingly used.

Safety and immunogenicity of Brucella abortus strain RB51 vaccine in pregnant cattle.

OBJECTIVE: To determine the safety and immunogenicity of Brucella abortus strain RB51 as a vaccine in pregnant cattle. ANIMALS: 12 Polled Hereford heifers obtained from a brucellosis-free herd and bred on site at 16 months of age to a brucellosis-free bull. PROCEDURE: Pregnant heifers were vaccinated at 6 months' gestation with 10(9) colony-forming units of B abortus strain RB51 (n = 5), 3 x 10(8) colony-forming units of B abortus strain 19 (n = 5), or sterile pyrogenfree saline solution (n = 2). Samples were periodically collected for serologic testing and lymphocyte blastogenesis assays. At full gestation, heifers were euthanatized and specimens were collected for bacteriologic culture, histologic analysis, and lymphocyte blastogenesis assay, using various antigenic stimuli. RESULTS: None of the strain RB51- or strain 19-vaccinates aborted or had gross or microscopic lesions at necropsy that were consistent with brucellosis. Maternal blood mononuclear cells from strain RB51- and strain 19-vaccinates had proliferative responses to gamma-irradiated strain RB51 and strain 19 that were greater than responses by cells from nonvaccinated controls. In contrast, maternal superficial cervical lymph node cells from strain 19-vaccinates had proliferative responses to gamma-irradiated strain RB51 or strain 19 bacteria greater than those of cells from RB51-vaccinates and nonvaccinated controls. None of the heifers vaccinated with strain RB51 developed antibodies detected by use of the standard tube agglutination test, but all developed antibodies to strain RB51 that reacted in a dot ELISA, using irradiated strain RB51 as antigen. CONCLUSIONS: Pregnant cattle can be safely vaccinated with strain RB51 without subsequent abortion or placentitis. Furthermore, strain RB51 is immunogenic in pregnant cattle, resulting in humoral and cell-mediated immune responses, but does not interfere with serologic diagnosis of field infections.

Control methods and thresholds of acceptability for antibrucella vaccines.

Protection against brucellosis involves both cellular and humoral effectors not yet fully appreciated. Living or killed vaccines can protect against the infection itself or only against abortion. For official controls, vaccines (or new procedures of vaccination) must first be characterized pharmacologically and tested for innocuity. Protection must be tested on natural hosts with a reference vaccine (S19 or Rev. 1) by the agreed method which reproduces the natural infection and measures immunity in toto. Control and vaccinated females are challenged by the conjunctival route at mid-pregnancy under standard conditions (strain, dose) to measure the resulting infection by bacteriological analysis of excretion at parturition and of infection in target organs at slaughter. Results are principally expressed by the infection rate which should be +/- 95% in the control group. In the new vaccine group the rate should be equivalent to, or lower than, the reference vaccine group. To be statistically valid, at least 30 animals per group are required. For routine controls, laboratory models using guinea pigs, not well standardized, inaccurate and expensive, have long been proposed. The mouse model, extensively studied and standardized, should now be preferred to the guinea pig model. In the mouse model, residual virulence of a living vaccine is estimated by the time required by 50% of the mice to eradicate the strain from their spleen (Recovery Time 50%). Immunogenicity is measured by the ability of mice to restrict their splenic infection after a virulent i.p. challenge at a dose (B. abortus 544; 2 x 10(5) cfu) chosen in order that all mice were still infected 15 days post challenge.(ABSTRACT TRUNCATED AT 250 WORDS)

Theoretical, practical and statistical basis for a general control method of activity for anti-Brucella vaccines.

Lots of five weeks old female CD-1 mice were vaccinated at several doses with living (B. abortus 19), killed whole cells or purified fractions vaccines. The doses were the number of bacterial cells injected per mouse, either viable (living vaccines), total (killed) or number calculated from the yield of extraction. The mice were intraperitoneally injected with a lyophilized standard challenge, 2 X 10(5) Brucella abortus 544, thirty days after vaccination. In two assays, B. melitensis H.38 was used as challenge at several doses. Responses to a good live vaccine were dose independent. Responses to killed whole cell and purified fractions were usually dose dependent. A reference lyophilized vaccine previously prepared and titrated would give the reference response to one unit. These data can be used to devise a general control method of activity for anti-Brucella vaccines. This was proposed and discussed in the preceding paper.

A laboratory reference vaccine to titrate immunogenic activity of antibrucella vaccines in mice.

The number of bacteria (CFU) in spleens of mice fifteen days after a standard intraperitoneal challenge of Brucella abortus, is dependent on vaccinal immune status of mice. From this observation, a control method of vaccinal activity was previously proposed, which classified the responses in relation to fixed reference values. A reference vaccine, a lyophilized formalin-killed bacterial cell suspension of B. melitensis strains H38 was prepared and titrated. From the dose response curve, the quantities giving reference values were calculated and expressed in a Unit system. This vaccine makes possible inter-laboratory comparisons of vaccinal activity, that can be expressed on the Units basis.